Regulatory T cells (Tregs) are one of the most important components in the immune suppressive networks in the tumor microenvironment. Tregs dampen tumor associated antigen (TAA)-specific T cell immunity and are thought to be the main obstacle tempering successful immunotherapy and active vaccination. Accumulating evidence indicates that tumor microenvironment controls the recruitment of Tregs, and Tregs interact with antigen presenting cells (APCs) and T cells to mediate immune suppression. Multiple modes of action of suppression are proposed in the literature to dissect their suppressive mechanisms. However, the cellular and molecular mechanisms controlling the survival, the phenotypic and functional integrity of Tregs in the human tumor microenvironment remain poorly understood. In this proposal we take human ovarian cancer as a typical research model to address these significant questions. Our specific aims are: Aim 1 is to test our hypothesis that functional CXCR4 on Tregs is induced by hypoxia in the tumor microenvironment. The HIF/CXCR4 pathway will be explored. Aim 2 is to test our hypothesis that Tregs survive hypoxia through interacting with tumor cells in the tumor microenvironment. The CXCR4/CXCL12 pathway will be explored. Aim 3 is to test the hypothesis that hypoxia regulates Treg functional stability and integrity in human ovarian cancer. The HIF/Notch pathway will be explored. Our proposal will provide a novel mechanism linking the HIF signaling pathway to Treg survival, phenotype and functional integrity in the human tumor microenvironment. Understanding this new molecular link and the relevance will shed significant light on human Treg biology and provide useful information for designing novel therapeutic strategies to treat patients with cancer. PUBLIC HEALTH RELEVANCE: It has been shown that tumor infiltrating effector T cells are positively and regulatory T cells (Tregs) are negatively associated with patient outcome in multiple human cancers. Tumor associated antigen (TAA)-specific T cell responses are induced by tumor immune therapy and vaccination in patients with cancer. However, the cellular and molecular mechanisms controlling the survival, the phenotypic and functional integrity of Tregs in the human tumor microenvironment remain poorly understood. Understanding the molecular circuitry that contributes to the maintenance and survival of Tregs in the tumor microenvironment will generate novel information on human Treg biology, and provide insight into new approaches in cancer therapy.